The limited success of antibiotic therapy in treating inhalational anthrax has motivated investigation of complementary therapeutic strategies that target the bacteria's secreted toxin. The metalloproteinase lethal factor (LF) is a critical component of anthrax toxin and an important potential target for small molecule drugs. The broad goal of the proposed work is to understand in detail interactions between LF and its host substrates, the MAP kinase kinases (MKKs), and to exploit these interactions for potential therapeutic benefit. We propose to identify small molecule LF inhibitors that target LF-MKK exosite interactions, which occur outside of the active site. To accomplish this we will conduct a high throughput screen using a full length MKK protein, rather than a short peptide, as a substrate. Hits from this primary screen will be put through a secondary screen to discard compounds that inhibit cleavage of a peptide substrate. Compounds targeting the LF exosite will be characterized for their efficacy as LF inhibitors in vitro and in cultured cells. Small molecules identified through screening will be used as structural probes to identify sites of interaction between LF and MKKs. This work will set the stage for development of these LF inhibitors into therapeutic leads. PUBLIC HEALTH RELEVANCE: Inhalational anthrax is a highly fatal disease of concern due to potential abuse of the causative bacteria as a biowarfare agent. Antibiotics are mostly ineffective against the disease due to a deadly toxin produced by the bacteria. We will identify molecules that block interactions between the toxin and proteins made by the infected host, with the aim of developing new drugs to neutralize the toxin.